Arenaviruses represent a rapidly growing group of emerging rodent-borne viruses. Highly pathogenic arenaviruses, Lassa (LASV), Junun (JUNV), and Machupo (MACV), are the most prevalent arenaviruses in the world that represent the emerging severe threat to the U.S. public health. The development of safe and efficacious vaccines against emerging pathogens with particular emphasis on multivalent strategy and advanced platform technology is one of the top priorities of NIH/NIAID. The main goal of this project is the development and feasibility testing of the first trivalent arenaviral vaccine against LASV, JUNV, and MACV. The trivalent vaccine is based on Medigen's VLPV (virus-like-particle-vector) technology. VLPVs comprise propagation-defective particles that encapsulate recombinant vector for delivery and expression in vivo of the full-length glycoprotein (GPC) genes derived from three arenaviruses. The proposed trivalent vaccine would protect populations from the world's major arenavirus threats, including containment of the outbreaks in non-endemic areas, as well as vaccination of medical workers, military personnel, travelers, and population in endemic areas. If successful, VLPV polyvalent vaccine platform can also be used as a generic approach for other infectious diseases. SPECIFIC AIM I: Design and Preparation of Trivalent VLPV Vaccine Expressing Arenaviral GPCs. The goal of this aim is (i) to prepare a tri-cistronic vector for expression of the full-length GPC genes derived from LASV, JUNV, and MACV;(ii) to prepare individual monocistronic VLPV vaccines for LASV, JUNV, and MACV;(iii) encapsulate tri- or monocistronic vectors into VLPVs for delivery and expression of arenaviral GPCs in vitro and in vivo;and (iv) to optimize and characterize expression levels, stability, and production yields of tri- and monocistronic VLPV vaccines. SPECIFIC AIM II: Preclinical Safety and Immunogenicity Evaluation of Trivalent Arenavirus Vaccines. Recombinant trivalent arenavirus vaccine prepared in Specific Aim I (tri-cistronic, individual, or blended combination of individual vaccines) will be used to evaluate their safety and immunogenicity in mice and guinea pigs including the ability to induce robust CD8+ T cell and neutralizing antibody (nAB) responses. PUBLIC HEALTH RELEVANCE: Highly pathogenic viruses, Lassa (LASV), Junun (JUNV), and Machupo (MACV) represent the world's most common arenaviruses and an emerging severe threat for the U.S. public health. The main goal of this project is the development and feasibility testing of the individual and trivalent vaccines against these arenaviruses. The successful trivalent vaccine can be used for the containment of disease outbreaks in non-endemic areas, as well as for vaccination of medical workers, military personnel, travelers, and population in endemic areas. If successful, Medigen's polyvalent vaccine platform can also be used as a generic approach for other infectious diseases.